Methods for the detection and accurate measurement of the presence and growth progression of various microorganisms are useful for a variety of purposes, including monitoring yields in the production of microorganisms in industrial fermentation process and the early detection of pathogenic microorganisms.
Several methods are known, an example of which is U.S. Pat. No. 5,523,214. In this reference, there is described a method for visually demonstrating the growth of microorganisms in broths or gels such as fungi, yeasts and bacteria including mycobacteria, M. tuberculosis, M. avium and M. bovis, non-fermenters, cocci, bacilli, coccobacilli and enterobacteria obtained from urine specimens, matter from wounds and abscesses, blood and sputum and bacterial growth in broths or gels. In this reference, it has been estimated that relatively rapidly growing mycobacteria require approximately one week to demonstrate growth, and relatively more slowly growing tuberculosis agents such as M. tuberculosis and M. bovis and M. avium, which are known to appear in AIDS patients, require at least eight to ten weeks of incubation. To detect growth in this method, a mixture of indicators methylene blue and resazurin is added to the substrate or environment with care taken that not enough of the mixture be added to be toxic to the microorganisms. The substrate is iron (III) salts mixed with K.sub.3 Fe(CN).sub.6, iron (II) salts mixed with K.sub.4 Fe(CN).sub.6 or sodium tungstate (Na.sub.2 WO.sub.4) As set forth in this reference, the mixture of indicators methylene blue and resazurin is said to demonstrate bacterial growth by changing color from blue to red more rapidly than resazurin alone. The method is also said to be improved by the addition of a redox stabilizer such as potassium hexacyanoferrate, K.sub.4 Fe(CN).sub.6.
As also related in this method, mixtures of inorganic salts of iron (III) such as NH.sub.4 Fe(SO.sub.4).sub.2 and K.sub.3 Fe(CN).sub.6, or iron (II) such as K.sub.4 Fe(CN).sub.6, or Na.sub.2 WO.sub.4 by itself are employed in culture media as redox indicators to demonstrate the growth of microorganisms.
Such a method is not commercially practical, however, as the amounts of redox indicators required to demonstrate microorganism growth are not consistently non-toxic, and/or require an inordinate amount of care to exclude toxic amounts to prevent false negative results. Such methods, as are all conventional methods, are not sufficiently sensitive to reduce the time required for demonstration of microbial growth from several weeks to a matter of days.